Isolation And Identification Of Microorganisms From Ancient Tomb Soil And Biosynthetic Study Of Natural Products Of Talaromyces Variavilis F34

Natural products play an important role in the history of human medicine because of their novel structure and good biological activity.However,due to the overuse of existing antibiotics,drug-resistant bacteria have had a serious impact on public health security.The development of new drugs is urgent.As major producers of natural products,microorganisms,especially fungi and actinomycetes,are important sources for the development of new drugs and lead molecules for new drugs.Microbes come from a wide range of sources,from the polar to the desert,from the soil to the deep sea,all distributed in huge numbers of microbial populations.These microorganisms have great potential to produce compounds with new structures.The microorganisms in this study were isolated from soil samples under the coffin lid of the tomb of Zeng Hou Bao in Jujube forest in Suizhou,Hubei Province.This project is divided into the following three parts.The first part is the isolation and purification of microorganisms.A total of 30 strains of fungi were isolated by the combined method of dilution-plate method and plate streaking,whose sequences were analyzed and identified as Talaromyces sp.,Aspergillus sp.Penicillium sp.,Cladosporium sp.,and Lecanicillium sp.Two actinomycetes were isolated by sucrose density gradient centrifuge method and identified by 16S rDNA as Nocardia sp and Pseudonocardia sp,respectively.The second part is the isolation and identification of secondary metabolites by structure-guided strategy.After the small-scale fermentation screening of the isolated fungi and actinomycetes,Talaromyces sp.F34 was futher selected for large-scale fermentation with rice medium.After extraction with EtoAc,the silica chromatography,sephadex-LH20 and high-performance liquid chromatography(HPLC)were combined to separate and purify the products,5 of the products were determined as kasanosin C,comazaphilone C,montagnuphilone A,skyrin,and orsellinic acid by 1H and 13C NMR.In the third part,the biosynthetic gene clusters of secondary metabolites of F34 were explored.After the whole genome sequencing of strain F34,the biosynthetic gene clusters of strain F34 was analyzed and predicted by antiSMASH,which gave rise to a total of 99 potential biosynthetic gene clusters.The two core PKS genes was located for the biosynthesis of the isolated azaphilones.In addition,the P450 enzyme RugG,which was responsible for dimerization reaction during the biosynthesis of skyrin,was heterologous expressed in yeast and the corresponding microsomes were purified for in vitro enzymatic reaction.In conclusion,firstly,the composition of microbial community in the special habitat of the tomb of Zeng Hou Bao in jujube forest was revealed,which provided ideas for preventing the biodegradation of ancient monuments.Secondly,it provided strain resources for the isolation of bioactive compounds.Thirdly,it provided genomic data and experimental data support for the biosynthetic pathway of azaphilones and skyrin in fungi.